Targeted Protease Compositions and Uses Related Thereto

ABSTRACT

This disclosure relates to targeted protease compositions and uses related thereto. In certain embodiments, the disclosure relates to nanoparticles wherein a targeting molecule is linked to the nanoparticle and wherein a catalytic domain of a protease is linked to the nanoparticle. In certain embodiments, the targeting molecule and the catalytic domain are within a single polypeptide sequence. In certain embodiments, the targeting molecule binds a molecule more highly expressed on cancer cells then non-cancerous cells, and the nanoparticles disclosed herein are used for the treatment of cancer by further attaching an anti-cancer agent to the nanoparticle or incorporating an anticancer agent within the nanoparticle.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application claims priority to U.S. Provisional Application No.61/913,989 filed Dec. 10, 2013 hereby incorporated by reference in itsentirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under Grant U01 CA151810-02 and R01 CA 154129A-01 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

BACKGROUND

Resistance to chemotherapy is a major challenge in treating cancer. Thetumor microenvironment includes several barriers to treatment. The tumorstroma promotes proliferation, invasion, metastasis, andchemoresistance. The enriched tumor stromal component and disorganizedvasculature of cancer tissues make it extremely difficult to deliver asufficient amount of therapeutic agents. Thus, there is a need toidentify improved therapeutic options.

Tumor targeted delivery can increase bioavailability of a drug to thetumor tissues while reducing systemic toxicity. Urokinase plasminogenactivator (uPA) is a serine protease that regulates multiple pathwaysinvolved in matrix degradation. Pancreatic cancer tissues have highlevels of uPAR expression in tumor cells, tumor endothelial cells, andtumor stromal fibroblasts and macrophages. In contrast, its expressionis not found in the normal pancreas or in pancreatic tissues withchronic pancreatitis.

Magnetic iron oxide nanoparticles (IONPs) are a biocompatible andbiodegradable nanoparticle. They can be used as molecular imaging probesfor targeted magnetic resonance imaging (MRI) and drug delivery. SeeYang et al., Receptor-targeted nanoparticles for in vivo imaging ofbreast cancer, Clin Cancer Res, 2009, 15(14):4722-32. Yang et al.,Molecular Imaging of Pancreatic Cancer in an Animal Tumor Model UsingTargeted Multifunctional Nanoparticles, Gastroenterology 2009, 136(5):1514-1525. Lee et al. report engineered urokinase plasminogen activatorreceptor (uPAR)-targeted magnetic iron oxide nanoparticles (IONPs)carrying chemotherapy drug gemcitabine (Gem) for targeted delivery intouPAR-expressing tumor and stromal cells. ACS Nano, 2013, 7(3):2078-89.See also WO 2008/073856.

Yoele et al., report a review of peptide nanomedicine as it relates tocancer treatment. Asian J Pharma & Clinical, 2013, Supp 2(6), 28. Seealso Zhang et al., Peptides in cancer nanomedicine: drug carriers,targeting ligands and protease substrates, J Control Release, 2012,159(1):2-13. Satpathy et al. report active targeting breast cancer cellsusing HER-2-affibody-conjugated nanoparticles. See Small, 2014,10(3):544-55.

Cho et al. report targeted delivery of siRNA-generating DNAnanocassettes using multifunctional nanoparticles. Small, 2013,9(11):1964-73. See also US Application Publication 2014/0105828.

References cited herein are not an admission of prior art.

SUMMARY

This disclosure relates to targeted protease compositions and usesrelated thereto. In certain embodiments, the disclosure relates tonanoparticles wherein a targeting molecule is linked to the nanoparticleand wherein a catalytic domain of a protease is linked to thenanoparticle. In certain embodiments, the targeting molecule and thecatalytic domain are within a single polypeptide sequence. In certainembodiments, the targeting molecule binds a molecule more highlyexpressed on cancer cells then non-cancerous cells, and thenanoparticles disclosed herein are used for the treatment of cancer byfurther attaching an anti-cancer agent to the nanoparticle orincorporating an anticancer agent within the nanoparticle.

In certain embodiments, the disclosure relates to compositionscomprising a conjugate comprising a targeting molecule and the catalyticdomain of a protease polypeptide and a fluorescent moiety is linked tothe protease conjugate or linked to the nanoparticle. Typically, theconjugate is linked to a nanoparticle. In some embodiments, thetargeting molecule is linked to the nanoparticle and the proteasepolypeptide is linked to the nanoparticle. In certain embodiments, atherapeutic agent, e.g., an anti-cancer agent, is linked to thenanoparticle.

In certain embodiments, the targeting molecule is a ligand, folic acid,receptor, tyrosine phosphate inhibitor, steroid, antibody, single chainfragment from the antibody of epidermal growth factor receptor(ScFvEGFR), antibody mimetic, HER-2 affibody, peptide fragment of thereceptor binding domain of urokinase plasminogen activator, humaninsulin like growth factor, or fragment thereof. In certain embodiments,the targeting molecule binds uPAR, EGFR, IGF-1R, or HER-2.

In certain embodiments, the protease is a matrix metalloprotease.

In certain embodiments, particles disclosed herein have two or moretargeting molecules and further comprise chemotherapy agents,anti-inflammatory agents, nucleic acids such as siRNAs or nucleic acidssuch as DNA encoding siRNA in operable combination with a promoter forcellular expression.

In certain embodiments, the disclosure relates to pharmaceuticalcompositions comprising compositions disclosed herein, and apharmaceutically acceptable excipient typically for use in the treatmentof cancer. In certain embodiments, the pharmaceutical compositionscomprise a second anti-cancer agent.

In certain embodiments, the disclosure relates to methods of treating orpreventing cancer comprising administering an effective amount of apharmaceutical composition disclosed herein to a subject in needthereof. In certain embodiments, the pharmaceutical composition isadministered in combination with a second anti-cancer agent.

In certain embodiments, the cancer is a stroma-rich cancer such aspancreatic, liver, triple negative breast, prostate, sarcoma, and lungcancer.

In certain embodiments, the compounds are administered or migrate intofibrotic drug resistant residual tumors in cancer patients who havefailed previous chemotherapy or Her-2 antibody targeted therapy.

In certain embodiment therapeutic particles disclosed herein areadministered to treat or prevent non-cancerous fibrotic tissues withactive stroma fibroblasts and macrophages, such as liver cirrhosis, andarteriosclerosis.

In certain embodiments, the disclosure relates to recombinantly producedpolypeptides disclosed herein. In certain embodiments, the polypeptidecomprises a human uPA sequence or segment thereof configured to bindurokinase plasminogen activator receptor and a catalytic domain of aprotease polypeptide. In certain embodiments, the disclosure relates torecombinant nucleic acids encoding the polypeptides disclosed herein. Incertain embodiments, the disclosure relates to recombinant vectorscomprising recombinant nucleic acids disclosed herein. In certainembodiments, the disclosure relates to expression systems configured toproduce recombinant polypeptides disclosed herein comprising vectorsdisclosed herein.

In certain embodiments, the disclosure relates to targeting uPA-ATF withonly the growth factor domain (amino acids 1-68) of ATF having improvedsolubility and higher uPAR-targeting efficiency. In certain embodiments,the disclosure relates to the fusion targeting ligand ATF68-MMP14 withdual uPAR targeting and MMP14 protease activity.

In certain embodiments, the disclosure relates to ATF_(MMP) conjugatednanoparticles comprising probes and therapeutic drugs wherein thenanoparticles are not limited to magnetic iron oxide nanoparticles.

In certain embodiments, the disclosure relates to targeted nanoparticlesconjugated with dual targeting ligands such as ATF_(MMP) and anothercancer cell targeted ligand, such as IGF-1, Her2 affibody/antibody, andEGF or single chain antibody against EGFR, to improve targeting andintra-tumoral cell drug delivery in heterogeneous tumor cells.

In certain embodiments, the disclosure relates to nanoparticlesdisclosed herein comprising near infrared dye, e.g., NIR-830, labeledATF_(MMP) as peptide targeted optical imaging probes for detection ofuPAR receptor expression in tumors, or NIR-830-ATF68-MMP14 conjugatednanoparticle imaging probe for multimodal imaging.

In certain embodiments, the disclosure relates to ATF_(MMP) single ordual targeted nanoparticles carrying chemotherapy drugs, small moleculardrugs, siRNAs or siRNA expressing DNA nanocassettes, nucleic acidsencoding RNA in operable combination with a promoter.

In certain embodiments, the disclosure contemplates administering aneffective amount of nanoparticle disclosed herein in an amount of 0.4 mgto 1.6 mg per kg body weight of the subject. In certain embodiments,dosing treatments of once per week for four to six weeks or more time asnecessary are contemplated.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows tumor growth curve during the drug treatment by differentnanoparticles (NPs). The data indicates MMP protease activity on thetargeted nanoparticles enhanced anti-tumor efficiency based on tumorvolume.

FIG. 1B shows data on for different treatments indicating tumor weightsafter the whole procedure of NP-treatment.

FIG. 1C shows data on iron accumulation in tumors after NP-treatmentusing chemical analysis of iron concentration in tumor tissue lysates.

FIG. 2 shows data on MMP14 activity on ATF_(MMP)-IONPs indicating animproved targeted delivery in a human breast cancer patient derivedxenograft (PDX) model. Tumor bearing mice received two i.v. injectionsof NIR-830-ATF_(MMP)-IONPs (400 picomol). Optical imaging was performed48 hrs following the injection. Only one of the three tumors in themouse received ATF-IONPs had strong optical signal. All four tumors inthe mouse received ATF_(MMP)-IONP had very strong optical signals. Anincrease in IONP accumulation was found in ATF_(MMP)-IONP treatedtumors, compared to ATF-IONP or MMP-IONP treated tumors.

FIG. 3 shows T2-weighted MRI detection of IONP accumulation of differentnanoparticles. Numbers are parentage of MRI signal decrease comparedwith pre-contrast images.

FIG. 4 shows data on a dual receptor targeted ATF_(MMP)-IGF-IONPindicating enhanced the intra-tumoral IONP delivery and distribution.Nude mice bearing human pancreatic PDX tumors receivedATF_(MMP)-IGF-IONP, ATF-IONP, IGF-IONP, or ATF_(MMP)-IONP (400 picomol)via the tail vein for two injections. Ex vivo optical imaging alsoshowed a high signal in the tumor treated with ATF_(MMP)-IGF-IONP. Dualreceptor targeting showed significant MRI signal decrease (40% dualtargeting with MMP) as compared from single targeting (˜10 to 18%).

FIG. 5A shows data on evaluation of the efficiency of targeted deliveryof nanoparticles by the combination of targeting to tumor cells andtumor stromal cells and MMP-14-mediated extracellular matrixdegradation. A mixture of NIR-830 dye labeled human ATF68 and mouseATF68 or human ATF68-MMP14_(CD) and mouse ATF68-MMP14_(CD) was used sothat the nanoparticles could target to uPAR expressing human cancercells and mouse-derived stromal cells. The tumor-bearing mice received200 pmol of different IONPs every two days for two injections. Opticalimaging was performed 48 hours following the last injection. A. Wholebody non-invasive NIR optical imaging and ex vivo organ imaging in aprimary human breast cancer model derived from a surgically resected andmulti-drug resistant triple negative breast cancer tissue. AlthoughATF68- or MMP-14_(CD) conjugated IONPs could target to tumors andproduce optical signals, there were marked differences in the signalintensity among multiple tumor lesions in the same mouse. It is possiblethat inter-tumoral heterogenicity in the tumor stromal structure andcellular components affected nanoparticle delivery into tumors.

FIG. 5B shows results for NIR-830 dye MMP-14_(CD) conjugated IONPs.

FIG. 5C shows results for the tumor-bearing mice that receivedNIR-830-ATF68-MMP-14_(CD)-IONPs. High levels of optical signals weredetected in all four tumor xenograft in a mouse, suggesting that atargeting ligand with MMP14CD has the ability to enhance intratumoralnanoparticle delivery and distribution. In ex vivo images, the numbersshowed the mean optical signals of tumors and normal organs. Lined areaswere size and location of tumors and normal organs, which showed that inthe tumors obtained from the mice that received ATF68- or MMP-14_(CD)conjugated IONPs, there were tumor areas without strong optical signals.Tumors from the mice that received NIR-830-ATF68-MMP-14_(CD)-IONPs hadstrong signals in almost all tumor areas.

FIG. 6 illustrates the fusion of ATF68 and the MMP14_(CD).

FIG. 7A illustrates ATF68-MMP14CD-IONPs

FIG. 7B illustrates NIR-830-ATF68-MMP14_(CD)-IONPs.

DETAILED DISCUSSION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of immunology, medicine, organic chemistry,biochemistry, molecular biology, pharmacology, and the like, which arewithin the skill of the art. Such techniques are explained fully in theliterature.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

In this specification and in the claims that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings unless a contrary intention is apparent.

As used herein, the term “combination with” when used to describeadministration with an additional treatment means that the agent may beadministered prior to, together with, or after the additional treatment,or a combination thereof.

As used herein, “subject” refers to any animal, typically a humanpatient, livestock, or domestic pet.

As used herein, the terms “prevent” and “preventing” include theprevention of the recurrence, spread or onset. It is not intended thatthe present disclosure be limited to complete prevention. In someembodiments, the onset is delayed, or the severity of the disease isreduced.

As used herein, the terms “treat” and “treating” are not limited to thecase where the subject (e.g., patient) is cured and the disease iseradicated. Rather, embodiments, of the present disclosure alsocontemplate treatment that merely reduces symptoms, and/or delaysdisease progression.

“Cancer” refers any of various cellular diseases with malignantneoplasms characterized by the proliferation of cells. It is notintended that the diseased cells must actually invade surrounding tissueand metastasize to new body sites. Cancer can involve any tissue of thebody and have many different forms in each body area. Within the contextof certain embodiments, whether “cancer is reduced” can be identified bya variety of diagnostic manners known to one skill in the art including,but not limited to, observation the reduction in size or number of tumormasses or if an increase of apoptosis of cancer cells observed, e.g., ifmore than a 5% increase in apoptosis of cancer cells is observed for asample particle compared to a control without the particle. It can alsobe identified by a change in relevant biomarker or gene expressionprofile, such as PSA for prostate cancer, HER2 for breast cancer, orothers.

The terms “nucleic acid sequence” refer to any nucleotide sequence(e.g., RNA or DNA), the manipulation of which may be deemed desirablefor any reason (e.g., treat disease, confer improved qualities, etc.),by one of ordinary skill in the art. Such nucleotide sequences include,but are not limited to, coding sequences of structural genes (e.g.,reporter genes, selection marker genes, oncogenes, drug resistancegenes, growth factors, etc.), and non-coding regulatory sequences whichdo not encode an mRNA or protein product (e.g., promoter sequence,polyadenylation sequence, termination sequence, enhancer sequence,etc.).

The terms “a nucleic acid sequence encoding” a specified polypeptiderefer to a nucleic acid sequence comprising the coding region of a geneor in other words the nucleic acid sequence which encodes a product. Thecoding region may be present in a cDNA, genomic DNA or RNA form. Whenpresent in a DNA form, the oligonucleotide may be single-stranded (i.e.,the sense strand) or double-stranded. Suitable control elements such asenhancers/promoters, splice junctions, polyadenylation signals, etc.,may be placed in close proximity to the coding region if needed topermit proper initiation of transcription and/or correct processing ofthe primary RNA transcript. Alternatively, the coding region utilized inthe expression vectors may contain endogenous enhancers, exogenouspromoters, splice junctions, intervening sequences, polyadenylationsignals, etc., or a combination of both endogenous and exogenous controlelements.

The term “recombinant nucleic acid” as used herein is defined as anucleic acid, e.g., DNA, produced by joining pieces from differentsources. The term “recombinant polypeptide” as used herein is defined asa polypeptide produced by using recombinant nucleic acids.

The terms “in operable combination,” “in operable order,” and “operablylinked” refer to the linkage of nucleic acid sequences in such a mannerthat a nucleic acid molecule capable of directing the transcription of agiven gene and/or the synthesis of a desired RNA or protein molecule isproduced. Transcriptional control signals in eukaryotes comprise“promoter” and “enhancer” elements. Promoters and enhancers consist ofshort arrays of DNA sequences that interact specifically with cellularproteins involved in transcription (see, for e.g., Maniatis, et al.(1987) Science 236:1237; herein incorporated by reference). Promoter andenhancer elements have been isolated from a variety of eukaryoticsources including genes in yeast, insect, mammalian and plant cells.Promoter and enhancer elements have also been isolated from viruses andanalogous control elements, such as promoters, are also found inprokaryotes. The selection of a particular promoter and enhancer dependson the cell type used to express the protein of interest. Someeukaryotic promoters and enhancers have a broad host range while othersare functional in a limited subset of cell types (for review, seeManiatis, et al. (1987), supra; herein incorporated by reference).

As used herein, the term “exogenous promoter” refers to a promoter inoperable combination with a coding region wherein the promoter is notthe promoter naturally associated with the coding region in the genomeof an organism. The promoter which is naturally associated or linked toa coding region in the genome is referred to as the “endogenouspromoter” for that coding region.

The term “expression” when used in reference to a nucleic acid sequencerefers to the process of converting genetic information encoded in agene into RNA (e.g., mRNA, rRNA, tRNA, shRNA, or miRNA) through“transcription” of the gene (i.e., via the enzymatic action of an RNApolymerase), and into protein where applicable (as when a gene encodes aprotein), through “translation” of mRNA.

“Expression vector” refers to a vector comprising a recombinant nucleicacid comprising expression control sequences operatively linked to anucleotide sequence to be expressed. An expression vector comprisessufficient cis-acting elements for expression in an expression system;other elements for expression can be supplied by the host cell or in anin vitro expression system. Expression vectors include all those knownin the art, such as cosmids, plasmids (e.g., naked or contained inliposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses,and adeno-associated viruses) that incorporate the recombinant nucleicacid.

Methods of introducing and expressing genes and producing and isolatingpolypeptides associated with the genes into a cell are known in the art.In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a nucleic acid into a host cell includecalcium phosphate precipitation, lipofection, particle bombardment,microinjection, electroporation, and the like.

Biological methods for introducing a nucleic acid of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a nucleic acid into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the nucleic acid, in order toconfirm the presence of the recombinant DNA sequence in the host cell, avariety of assays may be performed. Such assays include, for example,“molecular biological” assays well known to those of skill in the art,such as Southern and Northern blotting, reverse transcription polymerasechain reaction (RT-PCR) and PCR; “biochemical” assays, such as detectingthe presence or absence of a particular peptide, e.g., by immunologicalmeans (ELISAs and Western blots).

Cell-free translation systems are commercially available and manydifferent types and systems are well-known. Examples of cell-freesystems include prokaryotic lysates such as Escherichia coli lysates,and eukaryotic lysates such as wheat germ extracts, insect cell lysates,rabbit reticulocyte lysates, frog oocyte lysates and human cell lysates.Eukaryotic extracts or lysates may be preferred when the resultingprotein is glycosylated, phosphorylated or otherwise modified. Some ofthese extracts and lysates are available commercially (Promega; Madison,Wis.; Stratagene; La Jolla, Calif.; Amersham; Arlington Heights, Ill.;GIBCO/BRL; Grand Island, N.Y.). Membranous extracts, such as the caninepancreatic extracts containing microsomal membranes, are also availablewhich are useful for translating secretory proteins. Mixtures ofpurified translation factors have also been used successfully totranslate mRNA into protein as well as combinations of lysates orlysates supplemented with purified translation factors such asinitiation factor-1 (IF-1), IF-2, IF-3, elongation factor T (EF-Tu), ortermination factors.

An “amino acid” as used herein is meant to include both natural andsynthetic amino acids, and both D and L amino acids. “Standard aminoacid” means any of the twenty L-amino acids commonly found in naturallyoccurring peptides. “Nonstandard amino acid residues” means any aminoacid, other than the standard amino acids, regardless of whether it isprepared synthetically or derived from a natural source. As used herein,“synthetic amino acid” also encompasses chemically modified amino acids,including but not limited to salts, amino acid derivatives (such asamides), and substitutions. Amino acids contained within the peptides,and particularly at the carboxy- or amino-terminus, can be modified bymethylation, amidation, acetylation or substitution with other chemicalgroups which can change a peptide's circulating half life withoutadversely affecting activity of the peptide. Additionally, a disulfidelinkage may be present or absent in the peptides.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include non-natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

Sequence “identity” refers to the number of matching residues (expressedas a percentage) in a sequence alignment between two sequences of thealignment. As used herein, percentage identity of an alignment iscalculated using the number of identical positions divided by thegreater of the shortest sequence or the number of equivalent positionsexcluding overhangs wherein internal gaps are counted as an equivalentposition. For example the polypeptides GGGGGG and GGGGT have a sequenceidentity of 4 out of 5 or 80%. For example, the polypeptides GGGPPP andGGGAPPP have a sequence identity of 6 out of 7 or 85%.

Percent “similarity” is used to quantify the similarity between twosequences of the alignment. This method is identical to determining theidentity except that certain amino acids do not have to be identical tohave a match. Amino acids are classified as matches if they are among agroup with similar properties according to the following amino acidgroups: Aromatic—F Y W; hydrophobic-A V I L; Charged positive: R K H;Charged negative—D E; Polar—S T N Q.

The terms “variant” when used in reference to a polypeptide refer to anamino acid sequence that differs by one or more amino acids fromanother, usually related polypeptide. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties. One type of conservative amino acidsubstitutions refers to the interchangeability of residues havingsimilar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine. More rarely, a variant may have “non-conservative”changes (e.g., replacement of a glycine with a tryptophan). Similarminor variations may also include amino acid deletions or insertions (inother words, additions), or both. Guidance in determining which and howmany amino acid residues may be substituted, inserted or deleted withoutabolishing biological activity may be found using computer programs wellknown in the art, for example, DNAStar software. Variants can be testedin functional assays. Certain variants have less than 10%, andpreferably less than 5%, and still more preferably less than 2% changes(whether substitutions, deletions, and so on).

As used herein, the term “derivative” refers to a structurally similarcompound that retains sufficient functional attributes of the identifiedcompound. The derivative may be structurally similar because it islacking one or more atoms, substituted, a salt, in differenthydration/oxidation states, or because one or more atoms within themolecule are switched, such as, but not limited to, replacing an oxygenatom with a sulfur atom or replacing an amino group with a hydroxylgroup, replacing an aromatic CH with a nitrogen or sulfur. Thederivative may be a prodrug. Derivatives may be prepare by any varietyof synthetic methods or appropriate adaptations presented in syntheticor organic chemistry text books, such as those provide in March'sAdvanced Organic Chemistry: Reactions, Mechanisms, and Structure, Wiley,6th Edition (2007) Michael B. Smith or Domino Reactions in OrganicSynthesis, Wiley (2006) Lutz F. Tietze hereby incorporated by reference.

The term “substituted” refers to a molecule wherein at least onehydrogen atom is replaced with a substituent. When substituted, one ormore of the groups are “substituents.” The molecule may be multiplysubstituted. In the case of an oxo substituent (“═O”), two hydrogenatoms are replaced. Example substituents within this context may includehalogen, hydroxy, alkyl, alkoxy, alkanoyl, nitro, cyano, oxo,carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl,aryl, arylalkyl, heteroaryl, heteroarylalkyl, —NRaRb, —NRaC(═O)Rb,—NRaC(═O)NRaNRb, —NRaC(═O)ORb,—NRaSO2Rb, —C(═O)Ra, —C(═O)ORa,—C(═O)NRaRb, —OC(═O)NRaRb, —ORa, —SRa, —SORa,—S(═O)2Ra, —OS(═O)2Ra and—S(═O)2ORa. Ra and Rb in this context may be the same or different andindependently hydrogen, halogen hydroxyl, alkyl, alkoxy, alkanoyl,amino, alkylamino, dialkylamino, carbocyclyl, carbocycloalkyl,heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl,and heteroarylalkyl.

The term “optionally substituted,” as used herein, means thatsubstitution is optional and therefore it is possible for the designatedatom to be unsubstituted.

The term “nanoparticle” refers to a molecular conglomerate of aboutbetween 1 and 1000 nm in diameter. One more molecules or biomoleculeslinked to the nanoparticle typically refers to covalently attaching themolecules or biomolecules to a polymer based exterior or coating. Withincertain embodiment, the compositions and methods disclosed herein may beutilized with a variety of polymer coated particle such as, e.g.,quantum dots (QDs), metal particles, gold, silver, iron, and iron-oxidenanoparticles (IONPs).

IONPs are typically prepared with a mean particle diameter of 4-100 nm.IONPs may be prepared by aging a stoichiometric mixture of ferrous andferric salts in aqueous media under basic conditions. Control overparticle size (2-20 nm) and shape is provided by adjusting the pH, ionicstrength and the concentration of the growth solution. The nanoparticlescan be functionalized in situ using additives such as organic compounds(e.g. sodium citric) or polymers (e.g. dextran, polyvinyl alcohol).Other metals such as gold, cobalt, nickel, and manganese may beincorporated into the material.

High-temperature decomposition of Fe(CO)₅ in organic solvents is anotherway to prepare IONPs. Size (3-19 nm) can be varied using alternativetemperatures. Flame spray pyrolysis yields a range of magnetite,maghemite and wustite (FeO) particles IONPs. Iron precursor such asFe(CO)₅ and Fe(NO₃)₃ may be used. Flame spray pyrolysis can be used toproduce different nanoparticles (TiO₂, ZrO₂, silica, etc.) as well ashybrid particles (e.g. silica-IONPs).

Hydroxyl groups on the IONP provide a place for synthetic attachment ofdifferent functional groups. A range of chemistries can be used tostabilize metal nanoparticles, exploiting electrostatic, hydrophobic,chelating and covalent interactions. Carboxylic acid groups can interactwith the surface of IONPs by coordination processes. IONP synthesis inorganic solvents is typically conducted in oleic acid. A polymer coatingon the IONPs is preferred. Polymer attachment to the IONP surface by aninitiator fixed to the surface of the IONPs and the polymer is grownfrom the surface. Alternatively, a functional, pre-formed polymer isgrafted onto IONPs in situ. Copolymers with hydrophobic groups,carboxylic acid groups, polyethylene glycols, or amine groups arecontemplated. Polymers with a hydrophilic block and a hydrophobic blockare contemplated. See Yang et al., Clin Cancer Res, 2009 15:4722; Lin etal., Small, 2008, 4(3):334-341; Yu et a., Nanotechnology, 2006,17:4483-4487; Park et al., J. Mater. Chem., 2009, 19, 6412-6417; Boyeret al. NPG Asia Mater., 2010,2(1):23-30, Kim et al., Nanotechnology,2011, 22, 155101; all hereby incorporated by reference in theirentirety.

Linking molecules or polypeptides to the polymers can be accomplishedusing a variety of methods. Typically, primary amine containingcompounds and proteins may be conjugated to the carboxylic acid groupson the polymer mediated by a coupling reagent such as EDAC. See Yang etal., Small, 2009,5(2):235-43, hereby incorporated by reference in itsentirety. Other coupling methods are contemplated, e.g., poly-histidinesequence may be recombinantly incorporated into a polypeptide sequenceof the targeting molecule. A poly-histidine chelating agent may becoupled to the polymer surface, e.g., NTA-Ni. Mixing the histidinetagged polypeptide sequence attaches it to the polymer surface linkedthrough the chelating agent. The avidin/streptavidin-biotin interactionsmay be used, e.g., biotin may be coupled to the polymer surface andstreptavidin may be expressed as a fusion/chimera with the targetingmolecule.

Targeted Protease Compositions and Conjugated Nanoparticles

One of the major challenges in cancer treatment is that the majority ofdrug and drug candidates are impeded by the high-density stromal matrixthat surrounds cancer cells. It can take several days for an antibodytherapeutic to get into the tumor center. The major obstacle are: 1)abnormal vasculatures in tumors, both low vessel density in hypoxictumor region and immature, non-function blood vessels in tumor areaswith active angiogenesis; 2) high interstitial pressure in the tumor dueto inflammation and dysfunction in lymphatic drainage system; and 3)extensive tumor stroma and fibrosis in the tumor, e.g., over 50% ofpancreatic tumor mass is tumor stroma.

Although it is not intended that embodiments of the disclosure arelimited by any particular mechanism, it is believed that nanoparticlestargeted to tumor endothelial cells, tumor associated stromal fibroblastand macrophages will facilitate the nanoparticles navigating though thetumor endothelial cell layer and entering into tumor stroma.Interactions of the nanoparticles with tumor stromal cells allows thenanoparticles retaining in the stroma for extended periods of time andthe MMP-14_(CD) on the targeting ligand breaking downs the extracellularmatrix, which enable immigration of the nanoparticles in the tumorstroma to reach to cancer cells. Therefore, it is desirable to usetargeting nanoparticles containing MMP14_(CD) to overcome the tumorstromal barrier and improve efficiency of drug delivery into tumorcells.

Disclosed herein are nanoparticles linked with a protease MMP14_(CD),i.e., the catalytic domain of a broad extracellular matrixmetallopeptidase MT1-MMP. Experiments indicate that it functions tolocalized degradation of components of extracellular matrix (ECM) andshows substrate specificity on the stromal matrix by degrading stromalmatrix and breaking the physical barrier of drug delivery. Such anapproach has potential to increase drug delivery in many types of humancancer. It is contemplates to be particularly useful for the treatmentof pancreatic cancer and triple negative breast cancer since 30 to 50%of those human cancer tissues consist of tumor stoma. It is believedthey have the ability to migrate inside the tumor tissue improving drugdelivery into tumor cells and overcoming drug resistance by deliveringlarge amounts of anti-cancer agents into tumor cells.

In one example, magnetic iron oxide nanoparticles (IONPs) were used thatare targeted to urokinase plasminogen activator receptor (uPAR), whichis a cell surface receptor that is highly expressed in tumorendothelial, stromal fibroblasts and active macrophages, and cancercells. Methods for carrying various therapeutic agents in or on theIONPs are contemplated. Targeted optical and MR imaging, as well astargeted therapeutic effect in breast and pancreatic cancer animalmodels are contemplated.

In certain embodiments, the disclosure relates to compositionscomprising conjugates comprising a targeting molecule and a proteasepolypeptide. Typically, the conjugate is linked to a nanoparticle. Incertain embodiments, the targeting molecule is linked to thenanoparticle and the protease polypeptide is linked to the nanoparticle.

In certain embodiments the disclosure relates to a nanoparticlecomprising a recombinant fusion polypeptide comprising a human uPAsequence or segment thereof configured to bind urokinase plasminogenactivator receptor (uPAR) and a human metalloprotease sequence orsegment thereof configured to catalyze the degradation of anextracellular matrix protein such as, but not limited to, MMP14, MMP15,MMP16, and MMP17, metalloelastase (MMP12), collegenases (MMP1, MMP8,MMP13), gelatinases (MMP2, MMP9), stromelysins (MMP3, MMP10, MMP11),matrilysin (MMP1, MMP26), enamelysin (MMP20). Typically the catalyticdomain forming the active site comprises a zinc-binding motif of threehistidine residues found in the conserved sequence HEXXHXXGXXH (SEQ IDNO: 5) wherein X is individually at each occurrence any amino acid.

In certain embodiments, the disclosure relates to recombinantly producedpolypeptides comprising a human uPA sequence or segment thereofconfigured to bind urokinase plasminogen activator receptor whichcomprises SEQ ID NO: 2 or a polypeptide with greater than 30% sequenceidentity or similarity thereto and a catalytic domain of a proteasepolypeptide which comprises SEQ ID NO: 3 or a polypeptide with greaterthan 30% sequence identity or similarity thereto.

In certain embodiments, the disclosure relates to recombinantpolypeptides comprising or consisting of SEQ ID NO: 4 ,variants, orsequences with greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%sequence identity or similarity thereto.

In certain embodiments, the disclosure relates to recombinantpolypeptides comprising or consisting of a human uPA fragment sequenceof less than 135, 100, 90, 80, 70, 60, 50, 40, 30 amino acids, e.g., SEQID NO:2, variants, or sequences with greater than 30%, 40%, 50%, 60%,70%, 80%, 90%, or 95% sequence identity or similarity thereto and acatalytic domain of a human matrix metalloprotease, e.g., SEQ ID NO: 3,variants, or sequences with greater than 30%, 40%, 50%, 60%, 70%, 80%,90%, or 95% sequence identity or similarity thereto.

In certain embodiments, the fusion polypeptides disclosed herein maycontain one or more linking groups or amino acid spacers between thetargeting sequence and the protease sequence.

In certain embodiments, the core of the nanoparticle has a hydrodynamicsize of about between 10 nm to 100 nm, or 10 nm to 40 nm, or 20 nm to200 nm, or 5 nm to 500 nm in diameter. In certain embodiments, thenanoparticle comprises a core with an average size of about between 2and 200 nm, or 5 and 100 nm, or 10 and 50 nm. In certain embodiments,the core comprises iron, gold, silver, selenium, zinc, indium, copper,oxygen, sulfur, phosphorus, or combinations thereof. In certainembodiments, the core is a metal, combination of metals, asemiconductor, quantum dot, gold, silver, iron, or an iron oxideparticle. A 10 nm core size and hydrodynamic size of 20 to 30 nm wereused in certain embodiments.

In certain embodiments, the targeting molecule is a polypeptide ligand,growth factors, protein, antibody, or antibody fragment. In certainembodiments, the cell targeting molecule is a ligand that targets areceptor specifically expressed on tumor cells. In certain embodiments,the cell targeting molecule is human or mouse ATF (hATF or mATF) peptideor fragment thereof. In certain embodiments, the cell targeting moleculeis a tumor-targeting human monoclonal antibody or comprises asingle-chain variable fragment (scFv) thereof.

In certain embodiments, particles disclosed herein further comprising ananticancer agent.

In certain embodiments, the anticancer agent is conjugated to thepolymer coating through carboxylic acid groups. In certain embodiments,the anticancer agent is contained inside the polymer coating in the areaof hydrophobic groups.

In certain embodiments, a therapeutic agent is linked to or encapsulatedin the nanoparticle. In certain embodiments, therapeutic agent is ananticancer agent such as, but not limited to, temozolamide, gefitinib,erlotinib, docetaxel, cisplatin, 5-fluorouracil, gemcitabine, tegafur,raltitrexed, methotrexate, cytosine arabinoside, hydroxyurea,adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin,mitomycin-C, dactinomycin and mithramycin, vincristine, vinblastine,vindesine, vinorelbine taxol, taxotere, etoposide, teniposide,amsacrine, topotecan, camptothecin, bortezomib, anagrelide, tamoxifen,toremifene, raloxifene, droloxifene, iodoxyfene, fulvestrant,bicalutamide, flutamide, nilutamide, cyproterone, goserelin,leuprorelin, buserelin, megestrol, anastrozole, letrozole, vorazole,exemestane, finasteride, marimastat, trastuzumab, cetuximab, dasatinib,imatinib, bevacizumab, combretastatin, thalidomide, lenalidomide, orcombinations thereof.

In certain embodiments, nanoparticles and protease conjugates disclosedherein comprise a lysosomally degradable molecule linked to atherapeutic agent, e.g., wherein degradable molecule is the polypeptideGFLG (SEQ ID NO: 1) linked to the therapeutic agent. See Lee et al.report engineered urokinase plasminogen activator receptor(uPAR)-targeted magnetic iron oxide nanoparticles (IONPs) carryingchemotherapy drug gemcitabine (Gem) for targeted delivery intouPAR-expressing tumor and stromal cells. See ACS Nano, 2013,7(3):2078-89.

In certain embodiments, a near infrared dye can be conjugated to theprotease-linked targeting ligands and nanoparticles, providing opticalimaging capability. In certain embodiments, the dye is a(3,3-dimethyl-indol-1-ium-1-yl)-N-alkylsulfonate dye or salt thereofsuch as one of the formula:

or salts or derivatives thereof optionally substituted with one or moresubstituents.

In certain embodiments, a fluorescent moiety is linked to the proteaseconjugate or linked to the nanoparticle. In certain embodiments, thefluorescent moiety is a fluorescent dye, for example, NIR-830 dye, or afluorescent protein, for example, green fluorescent protein.

In certain embodiments, the targeting molecule binds uPAR, EGFR, orHER-2, PMSA, IGF-1R, folate receptor, transferrin receptor, MUC-1,integrin alphav beta3, cell surface nucleolin, CTLA-4, or VEGFR. Incertain embodiments, the targeting molecule is an antibody or antibodymimetic, or aptamer of a natural ligand thereof such as theamino-terminal fragment of uPA, EGF, or folic acid.

In certain embodiments, the disclosure relates to targetingmolecule-protease or ligand-protease fusion peptides, recombinantnucleic acids encoding the fusions polypeptides, recombinant vectorscomprising the nucleic acids, and expression systems producing thepolypeptides.

Methods of Use

In certain embodiments, the ligand-protease fusion peptide is conjugatedto nanoparticles for use as an efficient drug delivery system bybreaking the tumor stromal barrier. In one example, a receptor targetingligands was fused with a catalytic domain of a protease to produce arecombinant multifunctional targeting ligand with the ability oftargeting to cell surface receptor and digest extracellular matrix inthe tumor stroma. Examples of targeting ligands that bind to cellsurface receptors highly expressed in tumor cells or tissues include,but are not limited to, amino terminal fragments (ATF) of urokinaseplasminogen activator (uPA), IGF-1, EGF, Her-2 affibody, or other tumorhoming short peptides. Examples of proteases include but are not limitedto MMP-14, MMP1, MMP9, hyalurondase.

For example, the cDNA sequence of 68 amino acids of the receptor bindingdomain of uPA was fused with the catalytic domain of the MMP14.Recombinant targeting ligands with uPAR targeting ability and MMP14activity (ATF68-MMP14_(CD)) was produced. ATF68-MMP14_(CD) peptides wereconjugated to surface functionalized magnetic iron oxide nanoparticles(IONPs) or quantum dots. Target specificity and enhanced delivery of thenanoparticles have been demonstrated in vivo in animal tumor models. TheATF68-MMP14_(CD) peptides can be used to target nanoparticles carryingtherapeutic agents, such as doxorubicin, gemcitabine, and cisplatin.Although it is not intended that embodiments of this disclosure belimited by any particular mechanism, these protease-linked IONPs arebelieved to have the ability to break tumor stromal matrix and migrateinside tumor tissue to improve intratumoral nanoparticle distributionand potentially increase drug delivery into tumor cells.

The protease conjugates and targeted nanoparticles have broadapplications in targeted cancer therapy for many types of human cancersthat have extensive tumor stromal components, such as pancreatic, triplenegative breast, skin, head and neck, liver, sarcoma, lung, and prostatecancers.

In certain embodiments, the disclosure relates to methods of treating orpreventing cancer comprising administering an effective amount of apharmaceutical composition disclosed herein to a subject in needthereof. In certain embodiments, the pharmaceutical composition isadministered in combination with a second anti-cancer agent. In certainembodiments, the cancer is selected from Hodgkin and non-Hodgkinlymphoma, leukemia, cervical cancer, ovarian cancer, endometrial cancer,colon cancer, breast cancer, gastric cancer, lung cancer, renal cancer,ovarian cancer, pancreatic cancer, prostate cancer, glioblastoma, headcancer, neck cancer, thyroid cancer, and melanoma and non-melanoma skincancer.

In certain embodiments, the disclosure relates to methods of optical andMR imaging the nanoparticle in tumors. 3D-MRI enables monitoring ofintratumoral distribution of nanoparticles and tumor responses totherapeutics contained on or in the nanoparticles.

In certain embodiments, the disclosure relates to nanoparticles coatedwith amphiphilic polymers conjugated with molecules useful for targetingtumors, monitoring the location of the nanoparticles administered to asubject by MRI, and viewing the presence of the nanoparticles duringoptical image-guided surgery.

In certain embodiments, the disclosure relates to uses of particlesdisclosed herein as a theranostics. Theranostics are therapeutics withphysical properties that allows one to image molecular accumulation ofthe vehicles in vivo. Yang et al., WO/2007/018647, disclose binding andinternalization of tumor targeted-iron oxide particles using MRI. Seealso Yang et al., J. Biomed. Nanotechnol., 2008, 4, 439-449. Lammers etal., Biomaterials, 2009, 30(2):3466-3475, disclose the simultaneousdelivery of doxorubicin and gemcitabine to tumors in vivo usingpolymeric drug carriers.

In certain embodiments, the disclosure relates to methods comprisingpreoperatively administering a composition comprising nanoparticlesdisclosed herein and monitoring the location of the particles in thesubject by detecting it by MRI (magnetic resonance imaging) in an areaof the subject. In certain embodiments, the method further comprises thesteps of operating on the subject in the area of detected particles,imaging dye identified tumors binding the targeting molecule, andsurgically removing dye identified tumors or tissue.

In certain embodiments, the disclosure relates to methods comprisingpreoperatively administering cancer targeted nanoparticles conjugated todyes disclosed herein to a subject, optically imaging a tumor that bindthe nanoparticles intra-opertively, and removing tumors targeted withthe nanoparticles.

In certain embodiments, the disclosure contemplates imaging andeffecting cell lysis with nanoparticles using iron or iron oxide cores.See WO 2009/120702. In certain embodiments, the disclosure relates totargeting of cancer by local hyperthermia using composition and methodsdisclosed herein. Local hyperthermia can lead to induction of apoptosis,heat-shock protein release, and chemotherapy agent sensitivity of cancercells by exposure of cancer cells containing particles with an iron oriron oxide core to an alternating magnetic fields (<1000 kHz) that aresafe to normal cells.

In certain embodiments, the disclosure relates to methods for lysis of acancer cells comprising, administering to a subject nanoparticlesdisclosed herein and adjusting magnetic fields proximate the subject tocause cell lysis of cancer cell that absorb the particles afteradministration. Typically, the magnetic field is an oscillating magneticfield and the particles are heated to at least 37° C. in vivo typicallygreater than 41° C.

In certain embodiments, the disclosure relates to ATF_(MMP) single ordual targeted nanoparticles carrying chemotherapy drugs, small moleculardrugs, siRNAs or siRNA expressing DNA nanocassettes, nucleic acidsencoding RNA in operable combination with a promotor such as U6. See USApplication Publication 2014/0105828.

In certain embodiments, the nucleic acid is double stranded DNA havingbetween about 350 and 1500 base pairs or 400 and 1000 base pairs, or 550and 750 base pairs. In certain embodiments, the polymer coating containsmonomers with hydrophobic and hydrophilic groups. In certainembodiments, the hydrophilic groups are amine and carboxylic acidgroups. In certain embodiments, the nucleic acid is double stranded DNA.In certain embodiments, the RNA capable of RNA interference is RNA thatforms a hairpin. In certain embodiments, the RNA capable of RNAinterference is a short hairpin RNA. In certain embodiments, the RNAcapable of RNA interference comprises a survivin sequence of greaterthan 15, 16, 17, or 18 nucleotides. In certain embodiments, the promoteris U6 or H1. Human survivin mRNA sequence (also known as homo sapiensbaculoviral IAP repeat containing 5 (BIRC5) transcript variant 1) isACCESSION NM_001168.2, available athttp://www.ncbi.nlm.nih.gov/gene/332, hereby incorporated by reference.An example siRNA sense survivin sequenc is:5′-GAGGCTGGCTTCATCCACTGCCC-3′ (SEQ ID NO: 6);

In certain embodiments, the polymer coating is conjugated to a nucleicacid that encodes microRNA.

In certain embodiments, the core of the particle has a size of aboutbetween 5 nm and 100 nm, or 20 nm and 200 nm, or 5 nm and 500 nm indiameter. In certain embodiments, the core is a metal, combination ofmetals, a semiconductor, quantum dot, gold, silver, iron, or an ironoxide particle.

In certain embodiments, the cell targeting molecule is a polypeptide,ligand, receptor, protein, antibody, or antibody fragment. In certainembodiments, the cell targeting molecule is a ligand that targets areceptor specifically expressed on tumor cells. In certain embodiments,the cell targeting molecule is human ATF (hATF) peptide or fragmentthereof. In certain embodiments, the cell targeting molecule is atumor-targeting human monoclonal antibody or comprises a single-chainvariable fragment (scFv) thereof.

In certain embodiments, particles disclosed herein further comprising ananticancer agent.

In certain embodiments, the anticancer agent is conjugated to thepolymer coating through carboxylic acid groups. In certain embodiments,the anticancer agent is trapped inside the polymer coating in the areaof the hydrophobic groups.

In certain embodiments, the disclosure relates to methods of treating adisease or condition associated with an overexpression of a genecomprising administering particles disclosed herein with a polymercoating wherein the polymer is conjugated to a nucleic acid that encodesa RNA capable of RNA interference of the overexpressed gene in operablecombination with a promoter and wherein the polymer is conjugated to acell targeting molecule to a subject in need thereof in an effectiveamount.

In certain embodiments, the disease or condition is cancer and thesubject is diagnosed with cancer. In certain embodiments, the cancer isbreast or pancreatic cancer. In certain embodiments, the particles areadministered in combination with another anticancer agent.

Targeting Molecules

In certain embodiments, the targeting molecule binds a molecule morehighly expressed on cancer cells then non-cancerous cells. Proteaseconjugates and nanoparticles disclosed herein can be used for thetreatment of cancer by further attaching an anti-cancer agent, e.g., tothe protease conjugate or nanoparticle or incorporating an anticanceragent within the nanoparticle.

In certain embodiments, the targeting molecule is a ligand, growthfactor IGF-1, folate, receptor, inhibitor, steroid, antibody, singlechain fragment from the antibody of epidermal growth factor receptor(ScFvEGFR), antibody mimetic, HER-2 affibody, ATF of uPA, or fragmentthereof. In certain embodiments, the targeting molecule binds uPAR,EGFR, IGF-1R, or HER-2.

Urokinase plasminogen activator (uPA) is a serine protease thatregulates multiple pathways involved in matrix degradation, cellmotility, metastasis and angiogenesis. Interaction of the N-terminalgrowth factor domain of uPA with its cellular receptor (uPAR) results inthe conversion of the plasminogen to a serine protease. In addition toits role in activation of the process for degradation of extracellularmatrix, uPAR also activates α5β1 integrin and ERK signaling throughinteraction with EGFR and induces cell proliferation. Additionally, theuPA/uPAR complex can bind to the matrix protein, vitronectin, inassociation with transmembrane integrins, and activate intracellularsignaling molecules such as the protein kinases, promoting celladhesion, proliferation, and migration.

The cellular receptors for uPA (uPAR) are highly expressed in many humantumor cells, intratumoral fibroblasts and tumor endothelial cells. About54% of ductal carcinoma in situ (DCIS) and 73% of lobular carcinomatissues have over 50% of their cancer cells overexpressing uPAR. Anelevated level of uPAR is associated with tumor aggressiveness, thepresence of distant metastasis and poor prognosis. However, uPAR isundetectable in the majority of normal tissues or organs except for lowlevels expressed in macrophages, granulocytes, the uterus, thymus,kidney and spleen. Therefore, uPAR is an excellent molecular target forrecruiting nanoparticles to breast tumor sites.

The uPAR-binding domain of uPA is located to the amino-terminal fragment(ATF) of uPA. Studies have shown that ATF is a potent uPA bindingantagonist to its high affinity receptor (uPAR) at the surface of bothtumor and endothelial cells. Systemic or local delivery of anon-catalytic amino-terminal fragment (ATF) of uPA (residues 1-135)using an adenoviral vector or conjugated peptides prevents the formationof the uPA/uPAR complex, thus inhibiting tumor growth and angiogenesis.Yang et al., Clin Cancer Res., 2009,15(14):4722-32, discuss thepreparation of targeted iron oxide nanoparticle using a recombinantpeptide containing the amino-terminal fragment of urokinase-typeplasminogen activator (uPA) conjugated to magnetic iron oxidenanoparticles amino-terminal fragment conjugated-iron oxide nanoparticle(ATF-IONP). This nanoparticle targets uPA receptor, which isoverexpressed in breast cancer tissues.

The human epidermal growth factor receptor (EGFR) family includes EGFR(HER-1), EGFR-2 (HER-2), EGFR-3 (Her-3) and EGFR 4 (HER-4). The ligandsthat bind to EGFRs are divided into EGFR-like ligands such as EGF andTGF-α, and the heregulins. These ligands bind to EGFR monomers topromoter receptor dimerization and oligomerization that ultimatelyresults in the activation of the EGFR signaling pathway. This EGFRsignaling pathway plays a role in the regulation of cell proliferation,survival and differentiation.

Human breast carcinomas express high levels of the EGF receptors.Overexpression of this receptor has been associated with highlyaggressive breast cancer types and a poor response to therapeuticagents. Prior preclinical and clinical studies have shown that blockingthe EGFR via monoclonal antibodies or inhibition of EGFR tyrosine kinasewith small molecule inhibitors inhibits the growth of breast cancers andsensitize chemotherapy responses. Single-chain antibodies to EGFR thatcontain the specific EGFR binding region but lack the Fc region havebeen isolated from human scFv phage display libraries. Yang et al.,Small, 2009, 5(2):235-43, hereby incorporated by reference in itsentirety, discussed the preparation of EGFR targeted nanoparticlesconjugating a single-chain anti-EGFR antibody (ScFvEGFR).

Iron oxide nanoparticles conjugated to a purified antibody thatselectively binds to the epidermal growth factor receptor (EGFR)deletion mutant (EGFRvIII) present on human glioblastoma multiforme(GBM) cells were used for therapeutic targeting and MRI contrastenhancement of experimental glioblastoma, both in vitro and in vivo,after convection-enhanced delivery (CED). See Hadjipanayis et al.,Cancer Res, 2010, 70:6303, hereby incorporated by reference in itsentirety. In certain embodiments, the disclosure relates to targetingmolecule that is an antibody or antibody mimetic to EGFR or EGFRvIII foruse in treating glioblastoma multiforme.

In certain embodiments, the targeting molecule is a monoclonalantibody-610 that targets a surface antigen for use in treating coloncarcinoma. See Cerdan et al., Magn Reson Med, 1989, 12:151-63 1989,hereby incorporated by reference in its entirety.

In certain embodiments, the targeting molecule is an antibody tocarcinoembryonic antigen (CEA) that targets CEA for use in treatingcolon tumors. See Tiefenauer et al., Magn Reson Imaging, 1996,14:391-402, hereby incorporated by reference in its entirety.

In certain embodiments, the targeting molecule is a monoclonal antibodyL6 that targets a surface antigen for use in treating intracranialtumor. See Remsen et al., Am J Neuroradiol, 1996, 17:411-18, herebyincorporated by reference in its entirety.

In certain embodiments, the targeting molecule is transferrin thattargets transferrin receptor for use in treating carcinoma. See Kresseet al., Magn Reson Med, 1998, 40:236-42, hereby incorporated byreference in its entirety.

In certain embodiments, the targeting molecule is a monoclonal antibodyto Her-2, e.g., Herceptin, that targets Her-2 receptors for use intreating breast cancer. See Lee et al., Nat Med, 2007, 13:95-9; Artemovet al., Magn Reson Med, 2003, 49:403-8; and Huh et al., J Am Chem Soc,2005, 127:12387-91, all hereby incorporated by reference in theirentirety.

In certain embodiments, the targeting molecule is the EPPT peptide thattargets underglycosylated mucin-1 antigen (uMUC-1) for use in treatingbreast, colon, pancreas and lung cancer. See Moore et al., Cancer Res,2004, 64:1821-7, hereby incorporated by reference in its entirety.

In certain embodiments, the targeting molecule is folic acid thattargets folate receptor for use in treating mouth carcinoma and cervicalcancer, e.g., folic acid as co-targeting ligand with ATF-MMP14. See Chenet al., PDA J Pharm Sci Technol, 2007, 61:303-13; Sun et al., Small,2006, 4:372-9; and Sonvico et al., Bioconjug Chem, 2005, 16:1181-8, allhereby incorporated by reference in their entirety.

In certain embodiments, the targeting molecule is methotrexate thattargets folate receptor for use in treating cervical cancer. See Kohleret al., Langmuir, 2005, 21:8858-64, hereby incorporated by reference inits entirety.

In certain embodiments, the targeting molecule is a monoclonal antibodyA7 that targets colorectal tumor antigen for use in treating colorectalcarcinoma. See Toma et al., Br J Cancer, 2005, 93:131-6, herebyincorporated by reference in its entirety.

In certain embodiments, the targeting molecule is chlorotoxin peptidethat targets membrane-bound matrixmetalloproteinase-2 (MMP-2) for use intreating glioma. See Veiseh et al., Nano Lett, 2005, 5:1003-8, herebyincorporated by reference in its entirety.

In certain embodiments, the targeting molecule is F3 peptide thattargets surface-localized tumor vasculature for use in treating glioma.See Reddy et al., Clin Cancer Res, 2006, 12:6677-86, hereby incorporatedby reference in its entirety.

In certain embodiments, the targeting molecule is RGD or RGD4C thattargets integrins for use in treating melanoma and epidermoid carcinoma.See Zhang et al., Cancer Res, 2007, 67:1555-62 and Uchida et al., J AmChem Soc, 2006, 128:16626-33, both hereby incorporated by reference intheir entirety.

In certain embodiments, the targeting molecule is luteinizing hormonereleasing hormone (LHRH) that targets LHRH receptor for use in treatingbreast cancer. See Leuschner et al., Breast Cancer Res Treat, 2006,99:163-76, hereby incorporated by reference in its entirety.

In certain embodiments, the targeting molecule is CREKA peptide thattargets clotted plasma proteins for use in treating breast cancer. SeeSimberg et al., Proc Natl Acad Sci USA, 2007, 104:932-6, herebyincorporated by reference in its entirety.

In certain embodiments, the targeting molecule is an antibody toprostate specific membrane antigen (PSMA) that targets PSMA for use intreating prostate cancer. See Serda et al., Mol Imaging, 2007, 6:277-88,hereby incorporated by reference in its entirety.

In certain embodiments, the disclosure contemplates targeting moleculesin any of the disclosed embodiments that are antibodies or fragments orchimera, antibody mimetics, or aptamers or any molecular entity thatselectively binds receptors, proteins, or glycoproteins that are moreprevalent on cancer cells.

Numerous methods known to those skilled in the art are available forobtaining antibodies or antigen-binding fragments thereof. For example,antibodies can be produced using recombinant DNA methods (U.S. Pat. No.4,816,567). Monoclonal antibodies may also be produced by generation ofhybridomas in accordance with known methods. Hybridomas formed in thismanner are then screened using standard methods, such as enzyme-linkedimmunosorbent assay (ELISA) and surface plasmon resonance analysis, toidentify one or more hybridomas that produce an antibody thatspecifically binds with a specified antigen. Any form of the specifiedantigen may be used as the immunogen, e.g., recombinant antigen,naturally occurring forms, any variants or fragments thereof, as well asantigenic peptide thereof.

The modular structure of antibodies makes it possible to remove constantdomains in order to reduce size and still retain antigen bindingspecificity. Engineered antibody fragments allow one to create antibodylibraries. A single-chain antibody (scFv) is an antibody fragment wherethe variable domains of the heavy (V_(H)) and light chains (V_(L)) arecombined with a flexible polypeptide linker. The scFv and Fab fragmentsare both monovalent binders but they can be engineered into multivalentbinders to gain avidity effects. One exemplary method of makingantibodies and fragments includes screening protein expressionlibraries, e.g., phage or ribosome display libraries. Phage display isdescribed, for example, in U.S. Pat. No. 5,223,409.

In addition to the use of display libraries, the specified antigen canbe used to immunize a non-human animal, e.g., a rodent, e.g., a mouse,hamster, or rat. In one embodiment, the non-human animal includes atleast a part of a human immunoglobulin gene. For example, it is possibleto engineer mouse strains deficient in mouse antibody production withlarge fragments of the human Ig loci. Using the hybridoma technology,antigen-specific monoclonal antibodies derived from the genes with thedesired specificity may be produced and selected. U.S. Pat. No.7,064,244.

Humanized antibodies may also be produced, for example, using transgenicmice that express human heavy and light chain genes, but are incapableof expressing the endogenous mouse immunoglobulin heavy and light chaingenes. Winter describes an exemplary CDR-grafting method that may beused to prepare the humanized antibodies described herein (U.S. Pat. No.5,225,539). All of the CDRs of a particular human antibody may bereplaced with at least a portion of a non-human CDR, or only some of theCDRs may be replaced with non-human CDRs. It is only necessary toreplace the number of CDRs required for binding of the humanizedantibody to a predetermined antigen. Humanized antibodies or fragmentsthereof can be generated by replacing sequences of the Fv variabledomain that are not directly involved in antigen binding with equivalentsequences from human Fv variable domains. Exemplary methods forgenerating humanized antibodies or fragments thereof are provided byU.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No.5,693,762; U.S. Pat. No. 5,859,205; and U.S. Pat. No. 6,407,213. Thosemethods include isolating, manipulating, and expressing the nucleic acidsequences that encode all or part of immunoglobulin Fv variable domainsfrom at least one of a heavy or light chain. Such nucleic acids may beobtained from a hybridoma producing an antibody against a predeterminedtarget, as described above, as well as from other sources. Therecombinant DNA encoding the humanized antibody molecule can then becloned into an appropriate expression vector.

In certain embodiments, a humanized antibody is optimized by theintroduction of conservative substitutions, consensus sequencesubstitutions, germline substitutions and/or back mutations. An antibodyor fragment thereof may also be modified by specific deletion of human Tcell epitopes or “deimmunization” by the methods disclosed in U.S. Pat.No. 7,125,689 and U.S. Pat. No. 7,264,806. Briefly, the heavy and lightchain variable domains of an antibody can be analyzed for peptides thatbind to MHC Class II; these peptides represent potential T-cellepitopes. For detection of potential T-cell epitopes, a computermodeling approach termed “peptide threading” can be applied, and inaddition a database of human MHC class II binding peptides can besearched for motifs present in the VH and VL sequences. These motifsbind to any of the 18 major MHC class II DR allotypes, and thusconstitute potential T cell epitopes. Potential T-cell epitopes detectedcan be eliminated by substituting small numbers of amino acid residuesin the variable domains, or preferably, by single amino acidsubstitutions. Typically, conservative substitutions are made. Often,but not exclusively, an amino acid common to a position in humangermline antibody sequences may be used. The V BASE directory provides acomprehensive directory of human immunoglobulin variable regionsequences. These sequences can be used as a source of human sequence,e.g., for framework regions and CDRs. Consensus human framework regionscan also be used, e.g., as described in U.S. Pat. No. 6,300,064.

Antibody mimetics or engineered affinity proteins are polypeptide basedtargeting moieties that can specifically bind to targets but are notspecifically derived from antibody V_(H) and V_(L) sequences. Typically,a protein motif is recognized to be conserved among a number ofproteins. One can artificially create libraries of these polypeptideswith amino acid diversity and screen them for binding to targets throughphage, yeast, bacterial display systems, cell-free selections, andnon-display systems. See Gronwall & Stahl, J Biotechnology, 2009,140(3-4), 254-269, hereby incorporated by reference in its entirety.Antibody mimetics include affibody molecules, affilins, affitins,anticalins, avimers, darpins, fynomers, kunitz domain peptides, andmonobodies.

Affibody molecules are based on a protein domain derived fromstaphylococcal protein A (SPA). SPA protein domain denoted Z consists ofthree α-helices forming a bundle structure and binds the Fc protion ofhuman IgG1. A combinatorial library may be created by varying surfaceexposed residues involved in the native interaction with Fc. Affinityproteins can be isolated from the library by phage display selectiontechnology. Affibody to HER-2 has been described. See Orlova et al.,Cancer Res., 2007, 67:2178-2186, hereby incorporated by reference in itsentirety.

Monobodies, sometimes referred to as adnectins, are antibody mimicsbased on the scaffold of the fibronectin type III domain (FN3). SeeKoide et al., Methods Mol. Biol. 2007, 352: 95-109, hereby incorporatedby reference in its entirety. FN3 is a 10 kDa, β-sheet domain, thatresembles the V_(H) domain of an antibody with three distinct CDR-likeloops, but lack disulfide bonds. FN3 libraries with randomized loopshave successfully generated binders via phage display (M13 gene 3, gene8; T7), mRNA display, yeast display and yeast two-hybrid systems. SeeBloom & Calabro, Drug Discovery Today, 2009, 14(19-20):949-955, herebyincorporated by reference in its entirety.

Anticalins, sometimes referred to as lipocalins, are a group of proteinscharacterized by a structurally conserved rigid β-barrel structure andfour flexible loops. The variable loop structures form an entry to aligand-binding cavity. Several libraries have been constructed based onnatural human lipocalins, i.e., ApoD, NGAL, and Tlc. Anticalins havebeen generated for targeting the cytotoxic T-lymphocyte antigen-4(CTLA-4) and the vascular endothelial growth factor (VEGF). See Skerra,FEBS J., 275 (2008), pp. 2677-2683, hereby incorporated by reference inits entirety.

The ankyrin repeat (AR) protein is composed repeat domains consisting ofa β-turn followed by two α-helices. Natural ankyrin repeat proteinsnormally consist of four to six repeats. The ankyrin repeats form abasis for darpins (designed ankyrin repeat protein) which is a scaffoldcomprised of repeats of an artificial consensus ankyrin repeat domain.Combinatorial libraries have been created by randomizing residues in onerepeat domain. Different numbers of the generated repeat modules can beconnected together and flanked on each side by a capping repeat. Thedarpin libraries are typically denoted NxC, where N stands for theN-terminal capping unit, C stands for the C-terminal capping domain andx for the number of library repeat domains, typically between two tofour. A HER-2 binding darpin has been generated from a librarycontaining two randomized repeat domains (N2C library) and by anaffinity maturation strategy. Zahnd et al., J. Mol. Biol., 2007,369:1015-1028, hereby incorporated by reference in its entirety.

Aptamers refer to affinity binding molecules identified from randomproteins or nucleic acids libraries. Peptide aptamers have been selectedfrom random loop libraries displayed on TrxA. See Borghouts et al.,Expert Opin. Biol. Ther., 2005, 5:783-797, hereby incorporated byreference in its entirety. SELEX (“Systematic Evolution of Ligands byExponential Enrichment”) is a combinatorial chemistry technique forproducing oligonucleotides of either single-stranded DNA or RNA thatspecifically bind to a target. Standard details on generating nucleicacid aptamers can be found in U.S. Pat. No. 5,475,096, and U.S. Pat. No.5,270,163. The SELEX process provides a class of products which arereferred to as nucleic acid ligands or aptamers, which has the propertyof binding specifically to a desired target compound or molecule. EachSELEX-identified nucleic acid ligand is a specific ligand of a giventarget compound or molecule. The SELEX process is based on the fact thatnucleic acids have sufficient capacity for forming a variety of two- andthree-dimensional structures and sufficient chemical versatilityavailable within their monomers to act as ligands (form specific bindingpairs) with virtually any chemical compound, whether monomeric orpolymeric. Molecules of any size or composition can serve as targets.

Pharmaceutical Compositions

In certain embodiments, the disclosure relates to pharmaceuticalcompositions comprising particles disclosed herein and apharmaceutically acceptable excipient. In certain embodiments, thecomposition is a pill or in a capsule or the composition is liquidsolution such as an aqueous buffer, e.g., a pH of about 6.5, 7.0, or 7.5or between 6 and 8. In certain embodiments, the pharmaceuticallyacceptable excipient is selected from a filler, glidant, binder,disintegrant, lubricant, and saccharide. Optionally, the pharmaceuticalcomposition further comprises a second anticancer agent.

Compositions suitable for parenteral injection may comprisephysiologically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions, and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents solventsor vehicles include water, ethanol, polyols (propylene glycol,polyethylene glycol, glycerol, and the like), suitable mixtures thereof,vegetable (such as olive oil, sesame oil and viscoleo) and injectableorganic esters such as ethyl oleate.

Prevention of the action of microorganisms may be controlled by additionof any of various antibacterial and antifungal agents, example,parabens, chlorobutanol, phenol, sorbic acid, and the like. It may alsobe desirable to include isotonic agents, for example sugars, sodiumchloride, and the like. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Solid dosage forms for oral administration include capsules, tablets,pills, powders and granules. In such solid dosage forms, the particlesmay be admixed with at least one inert customary excipient (or carrier)such as sodium citrate or dicalcium phosphate or: (a) fillers orextenders, as for example, starches, lactose, sucrose, glucose, mannitoland silicic acid, (b) binders, as for example, carboxymethylcellulose,alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c)humectants, as for example, glycerol (d) disintegrating agents, as forexample, agar-agar, calcium carbonate, potato or tapioca starch, alginicacid, certain complex silicates, and sodium carbonate, (e) solutionretarders, as for example paraffin, (f) absorption accelerators, as forexample, quaternary ammonium compounds, (g) wetting agents, as forexample cetyl alcohol, and glycerol monostearate, (h) adsorbents, as forexample, kaolin and bentonite, and (i) lubricants, as for example, talc,calcium stearate, magnesium stearate, solid polyethylene glycols, sodiumlauryl sulfate, or mixtures thereof. In the case of capsules, tablets,and pills, the dosage forms may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar and as high molecular weight polyethylene glycols, and thelike.

Solid dosage forms such as tablets, capsules, pills, and granules can beprepared with coatings and shells, such as enteric coatings and otherswell known in the art. They may contain opacifying agents, and can alsobe of such composition that they release the particles in a certain partof the intestinal tract in a delayed manner. Examples of embeddingcompositions which can be used are polymeric substances and waxes.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Inaddition to the particles, the liquid dosage forms may contain inertdiluents commonly used in the art, such as water or other solvents,solubilizing agents and emulsifiers, for example, ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils, in particular, cottonseed oil, groundnut oil,corn germ oil, olive oil, viscoleo, castor oil and sesame oil, glycerol,tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters ofsorbitan or mixtures of these substances, and the like.

Besides such inert diluents, the composition can also include adjuvants,such as wetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents. Suspensions, in addition to theparticles, may contain suspending agents, as for example, ethoxylatediso-stearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite agar-agarand tragacanth, or mixtures of these substances, and the like.

Pharmaceutical compositions typically comprise an effective amount ofparticles and a suitable pharmaceutical acceptable carrier. Thepreparations can be prepared in a manner known per se, which usuallyinvolves mixing the particles according to the disclosure with the oneor more pharmaceutically acceptable carriers, and, if desired, incombination with other pharmaceutical active compounds, when necessaryunder aseptic conditions. Reference is made to U.S. Pat. No. 6,372,778,U.S. Pat. No. 6,369,086, U.S. Pat. No. 6,369,087 and U.S. Pat. No.6,372,733 and the further references mentioned above, as well as to thestandard handbooks, such as the latest edition of Remington'sPharmaceutical Sciences.

The pharmaceutical preparations of the disclosure are preferably in aunit dosage form, and can be suitably packaged, for example in a box,blister, vial, bottle, sachet, ampoule or in any other suitablesingle-dose or multi-dose holder or container (which can be properlylabeled); optionally with one or more leaflets containing productinformation and/or instructions for use. Generally, such unit dosageswill contain between 1 and 1000 mg, and usually between 5 and 500 mg, ofthe particles of the disclosure e.g., about 10, 25, 50, 100, 200, 300 or400 mg per unit dosage.

The particles can be administered by a variety of routes including theoral, ocular, rectal, transdermal, subcutaneous, intravenous,intramuscular or intranasal routes, depending mainly on the specificpreparation used. The particles will generally be administered in an“effective amount,” by which it is meant any amount of particles that,upon suitable administration, is sufficient to achieve the desiredtherapeutic or prophylactic effect in the subject to which it isadministered. Usually, depending on the condition to be prevented ortreated and the route of administration, such an effective amount willusually be between 0.01 to 1000 mg per kilogram body weight of thesubject per day, more often between 0.1 and 500 mg, such as between 1and 250 mg, for example about 5, 10, 20, 50, 100, 150, 200 or 250 mg,per kilogram body weight of the subject per day, which can beadministered as a single daily dose, divided over one or more dailydoses. The amount(s) to be administered, the route of administration andthe further treatment regimen can be determined by the treatingclinician, depending on factors such as the age, gender and generalcondition of the subject and the nature and severity of thedisease/symptoms to be treated.

Formulations containing particles described herein can be prepared usinga pharmaceutically acceptable carrier composed of materials that areconsidered safe and effective and can be administered to an individualwithout causing undesirable biological side effects or unwantedinteractions. The carrier is all components present in thepharmaceutical formulation other than the active ingredient oringredients. As generally used herein “carrier” includes, but is notlimited to, diluents, binders, lubricants, disintegrators, fillers, pHmodifying agents, preservatives, antioxidants, solubility enhancers, andcoating compositions.

Carrier also includes all components of the coating composition whichcan include plasticizers, pigments, colorants, stabilizing agents, andglidants. Delayed release, extended release, and/or pulsatile releasedosage formulations can be prepared as described in standard referencessuch as “Pharmaceutical dosage form tablets,” eds. Liberman et. al. (NewYork, Marcel Dekker, Inc., 1989), “Remington—The science and practice ofpharmacy,” 20th ed., Lippincott Williams & Wilkins, Baltimore, Md.,2000, and “Pharmaceutical dosage forms and drug delivery systems,” 6thEdition, Ansel et al., (Media, PA: Williams and Wilkins, 1995). Thesereferences provide information on carriers, materials, equipment andprocess for preparing tablets and capsules and delayed release dosageforms of tablets, capsules, and granules.

Examples of suitable coating materials include, but are not limited to,cellulose polymers such as cellulose acetate phthalate, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulosephthalate and hydroxypropyl methylcellulose acetate succinate; polyvinylacetate phthalate, acrylic acid polymers and copolymers, and methacrylicresins that are commercially available under the trade name EUDRAGIT®(Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

Diluents, also referred to as “fillers,” are typically necessary toincrease the bulk of a solid dosage form so that a practical size isprovided for compression of tablets or formation of beads and granules.Suitable diluents include, but are not limited to, dicalcium phosphatedihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol,cellulose, microcrystalline cellulose, kaolin, sodium chloride, drystarch, hydrolyzed starches, pregelatinized starch, silicone dioxide,titanium oxide, magnesium aluminum silicate and powdered sugar.

Binders are used to impart cohesive qualities to a solid dosageformulation, and thus ensure that a tablet or bead or granule remainsintact after the formation of the dosage forms. Suitable bindermaterials include, but are not limited to, starch, pregelatinizedstarch, gelatin, sugars (including sucrose, glucose, dextrose, lactoseand sorbitol), polyethylene glycol, waxes, natural and synthetic gumssuch as acacia, tragacanth, sodium alginate, cellulose, includinghydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose,and veegum, and synthetic polymers such as acrylic acid and methacrylicacid copolymers, methacrylic acid copolymers, methyl methacrylatecopolymers, aminoalkyl methacrylate copolymers, polyacrylicacid/polymethacrylic acid and polyvinylpyrrolidone.

Lubricants are used to facilitate tablet manufacture. Examples ofsuitable lubricants include, but are not limited to, magnesium stearate,calcium stearate, stearic acid, glycerol behenate, polyethylene glycol,talc, and mineral oil.

Disintegrants are used to facilitate dosage form disintegration or“breakup” after administration, and generally include, but are notlimited to, starch, sodium starch glycolate, sodium carboxymethylstarch, sodium carboxymethylcellulose, hydroxypropyl cellulose,pregelatinized starch, clays, cellulose, alginine, gums or cross linkedpolymers, such as cross-linked PVP (Polyplasdone XL from GAF ChemicalCorp).

Stabilizers are used to inhibit or retard decomposition reactions whichinclude, by way of example, oxidative reactions.

EXAMPLES

Protease-Linked Migrating IONPs with uPAR-Targeting Fragments ATF39 andATF68

The amino terminal fragment (ATF, 135 aa) of uPA was used as a targetligand to develop uPAR-targeted theranostic IONPs. The yield and purityof recombinant ATF from bacterial cultures have been less than optimaldue to the formation of undesirable aggregates. By removing of thehydrophobic amino acids that promote the formation of aggregates, uPARtargeting ligands (39 and 68 aa) with receptor-binding peptide regionswere engineered as improvements. The ATF39 and ATF68 peptides can beproduced in high yield (8.6 mg/L of bacterial culture, compared to 0.5mg/L for 135 aa), increased purity and better solubility. Targetspecificity of the new ligand-conjugated IONPs was demonstrated in ahuman breast cancer PDX model derived from surgically resected andmultidrug resistant breast cancer tissues obtained triple negativebreast cancer patients.

The uPA-ATF with only the growth factor domain (ATF68) has improvedphysiological solubility and higher uPAR-targeting efficiency. Incomparison with the full length ATF (135 aa), this short uPAR targetedligand is an improved candidate to engineer the fusion targeting ligandATF68-MMP14 with dual uPAR targeting and MMP14 protease activity.

MMP14_(CD) IONP with uPAR-Targeting

Extensive tumor stroma in triple negative breast cancers creates aphysical barrier for drug delivery. To address the challenge of deliveryof the IONPs into cancer cells, fusion proteins of uPAR targetingligands and catalytic domain of MMP 14_(CD) were developed to facilitateactive targeting tumor and stromal and digesting of extracellular matrixproteins. The cDNA gene sequences of 39, 68 or 135 aa of human or mouseATF were cloned at the 5 ‘-end of the catalytic domain of the MMP14_(CD)gene. ATF68-MMP14_(CD) protein was produced in the bacterial expressionsystem.

Human uPA-ATF68-MMP14_(CD) protein sequence (SEQ ID NO: 4) underlinedportion is the AFT68 segment, amino acids 2-69 (SEQ ID NO:2) and segmentafter, i.e., amino acids 71-246 including the double underlined zincbinding domain, is the MMP14_(CD) (SEQ ID NO: 3).

(SEQ ID NO: 4) MSNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGAPIQGLKWQHNEITFCIQNYTPKVGEYATYEAIRKAFRVWESATPLRFREVPYAYIREGHEKQADIMIFFAEGFHGDSTPFDGEGGFLAHAYFPGPNIGGDTHFDSAEPWTVRNEDLNGNDIFLVAVHELGHALGLEHSSDPSAIMAPFYQWMDTENFVLPDDDRRGIQQLYGGESGThe protein was linked to IONPs comprising NIR-830 dye as reported inSatpathy et al., Small, 2014, 10(3):544-55, Xi et al J. Biophotonics 7,No. 6, 401-409 (2014), and Lee et al., ACS Nano, 2013, 7(3):2078-89.After labeled with near-infrared (NIR) dye on the thiol-group ofCysteine residues, ATF_(MMP) with his-tag at C-termini was thenconjugated to 10 nm core size IONP through two methods: 1) affinitybinding to a surface polymer with NTA-Cu functional groups and 2) directconjugation of amine groups of targeting peptides with carboxyl groupson the polymer surface via an amide bond. The polymer coated IONPs wereobtained from Ocean Nanotech LLC, San Diego, Calif. PEG-polymer coatedIONP has a hydrodynamic size of about 12 nm, and then increases to beabout 19 nm after conjugating with ATF is conjugated. ATF-MMP conjugatedIONP has a hydrodynamic size of about 24 nm.

To determine if conjugation to nanoparticle affects its proteolysisactivity, MMP14 activity of unconjugated ATF-MMP and ATF-MMP-IONP wasmeasured using a fluoregenic MMP14 substrate. About 80% of theproteolysis activity of ATF-MMP remained after being conjugated to thenanoparticle. It is believed that delivery of ATFMMP-IONP tointerstitial binding to stromal cells leads to protease activity onextracellular matrix to break intensive collagen fibers, resulting tinyholes that are permeable for targeted IONPs to migrate through stroma toreach tumor cells and deliver drugs into cancer cells.

Due to the presence of species specificity in the binding of ATF touPAR, a fusion protein was produced with mouse ATF68 and MMP14_(CD).Such as mouse uPAR targeted ligand is very useful for the evaluation ofthe effect of targeting tumor endothelial and stromal cells as well astumor cells on intratumoral nanoparticle delivery and distribution sinceall tumor stroma components originate from the mouse. Systemic deliveryof NIR-830-mATF68-MMP14_(CD)-IONPs led to accumulation of the IONPs inthe tumors in 4T1 mouse mammary tumor model. Moreover, stronger opticalsignals were detected in the tumors of the mice that receivedNIR-830-ATF68-MMP14_(CD)-IONPs than that of NIR-830-ATF68-IONPs orNIR-830-MMP14_(CD), indicating that a higher level of the IONPsaccumulate in the tumor.

The effect of a significant increase on intratumoral delivery andimproved intratumoral distribution of the nanoparticles was furtherconfirmed in a human breast cancer patient-derived xenograft model. Inthis study, a mixture of human ATF68 and mouse ATF68 to conjugate theIONPs were used so that they can efficiently target to both human tumorcells and mouse derived tumor stromal cells. Following systemic deliveryof 200 pmol of NIR-830-human +mouse ATF68-MMP14_(CD)-IONPs every 48hours for two injections, non-invasive optical showed markedly higherlevel of the nanoparticle accumulation in the tumor of the mice thatreceived NIR-830-hATF68-MMP14_(CD)-IONPs, compared withNIR-830-hATF68-IONPs with MMP14_(CD), or NIR-830-MMP14_(CD)-IONPs.MMP14_(CD) itself has the ability of targeted delivery of IONPs intotumors, most likely due to its membrane-binding property. However,histological examination of tumor tissue sections using Prussian bluestaining showed that the level of accumulation in the tumors receivedhATF68-MMP14_(CD)-IONP was significantly higher than that of eitherNIR-830-hATF68-IONPs, or NIR-830-MMP14_(CD)-IONPs. Furthermore, muchmore ATF68_(MMP)14_(CD) targeted IONPs were found in the tumor centralareas, compared to hATF68-IONPs without MMP14_(CD) or IONPs conjugatedwith MMP14_(CD) alone (FIG. 5A-C).

The ATF_(MMP) conjugated nanoparticle acts as an imaging probe and drugcarrier. The nanoparticles are not limited to magnetic iron oxidenanoparticles. These targeted nanoparticles may be conjugated with othertargeting ligands, e.g., ATF_(MMP) and another cancer cell targetedligand, such as IGF-1, Her2 affibody, and EGF or single chain antibodyagainst EGFR, to improve targeting and intratumoral cell drug deliveryin heterogeneous tumor cells.

In certain embodiments, the disclosure contemplates further conjugationwith a near infrared dye, NIR-830, labeled ATF_(MMP) as peptide targetedoptical imaging probes for detection of uPAR receptor expression intumors, or NIR-830-ATF68-MMP14 conjugated nanoparticle imaging probe formultimodal imaging.

In certain embodiments, simultaneous conjugation of separate targetingpeptides and separate MMP catalytic domain directly onto a nanoparticleis also contemplated. However, it is more desirable to have both in thesame polypeptide to save the conjugation sites on the surface ofnanoparticles for adding therapeutic agents linked to the surface.

Enhanced Nanoparticle Drug Delivery Led to Increased Anti-Tumor EffectsFollowing Systemic Delivery ATF_(MMP)-IONP Carrying Dox in an OrthotopicHuman Breast PDX Cancer Model

Doxorubicin, a commonly used chemotherapy drug, was encapsulated intoIONPs as reported in Yang et al., J Biomed Nanotechnol. Dec 1, 2008;4(4): 439-449. Following systemic delivery of 5 mg/Kg Dox equivalentdose of various IONPs once week for six treatments, significant tumorgrowth inhibition was detected in ATF_(MMP)-IONP-Dox treated tumorscompared with control, free Dox, nontargeted MMP-IONP-Dox andATF-IONP-Dox treated groups (FIGS. 1A-1C). Although non-targetedMMP-IONP-Dox have been shown to be able to deliver into tumors vialeaking tumor vessels and high collagen substrate in the tumor, mousegroup treated with MMP-NP-Dox did not show any therapeutic effect andsometime, had increased tumor growth compared to the controlno-treatment group. Therefore, ATF mediated targeting andinternalization of nanoparticle-drug is important for effective cancertherapy. Increased IONP-drug delivery was determined by chemicalanalysis of iron concentration in tumor lysates. The highest ironcontent was also detected in ATF_(MMP)-IONP-Dox treated tumors comparedto ATF-IONP treated tumors. Similar results were obtained from humanbreast cancer PDX models derived from two patients.

Multiplex receptor targeting using ATF_(MMP) and a tumor cell surfacereceptor targeting ligand further enhances intratumoral nanoparticledelivery uPAR is expressed in invasive and aggressive tumor cells, andits expression on tumor cells is heterogeneous. To ensure effectivereceptor mediated internalization of nanoparticle drugs into all tumorcells, dual receptor targeted IONP were prepared by conjugating bothNIR-830-dye labeled ATF_(MMP) and insulin growth factor 1 (IGF-1) toIONP. Insulin growth factor receptor 1 is expressed uniformly in mosttumor cells. It is also expressed at intermediate to high level in tumorstromal fibroblasts and macrophages. However, it is not expressed intumor vessel endothelial cells. The combination of targeting bothreceptors should facilitate nanoparticle drugs across tumor vessels,penetrating through tumor stromal barriers, and efficiently deliveringnanoparticle-drug into tumor cells. Supporting our hypothesis,

Systemic administration of dual receptor targeted IONPs into micebearing human pancreatic PDX tumors had a significantly higher level ofIONPs in the tumors than that of tumors received single receptortargeted IGF-IONP, ATF-IONP, or ATF_(MMP)-IONP. MRI result showed 40% T2signal decrease in the tumor of the mice that received dual targetedIONPs while the tumors of the mice that received single targeted IONPshad 14 to 22% T2 signal decrease. Ex vivo optical imaging of the excisedpancreatic tumors further confirmed the increased nanoparticle delivery.Histological analysis using Prussian blue staining revealed a high levelof IONPs in both orthotopic pancreatic cancer and liver metastasis. Allthree receptor targeted IONPs were found in tumor metastases in theliver, spleen and peritoneal cavity.

Conjugation of the ATF_(MMP) targeting ligands to nanoparticles resultedin nanoparticle imaging probes and drug carriers with improvedextravasation by targeting uPAR expressing tumor endothelial cells;improved binding to tumor stromal fibroblasts and macrophages thatincreases retention of the nanoparticles in tumor tissues; improveddigestion of stromal matrix proteins enabling penetration of thenanoparticles through tumor stroma to reach tumor cells, and improvedreceptor-mediated internalization of nanoparticle-drug that promotesintratumoral drug delivery.

What is claimed is:
 1. A composition comprising a conjugate comprising atargeting molecule and the catalytic domain of a protease polypeptide.2. The composition of claim 1, wherein the conjugate is linked to ananoparticle.
 3. The composition of claim 2, wherein the targetingmolecule is linked to the nanoparticle and the protease polypeptide islinked to the nanoparticle.
 4. The composition of claim 1, wherein atherapeutic agent is linked to the nanoparticle.
 5. The composition ofclaim 4, wherein the therapeutic agent is an anticancer agent.
 6. Thecomposition of claim 1, wherein a fluorescent moiety is linked to theprotease conjugate or linked to the nanoparticle.
 7. The composition ofclaim 1, wherein the targeting molecule comprises ATF₆₈ of uPA (SEQ IDNO: 2) and human insulin like growth factor (IGF-1).
 8. The compositionof claim 1, wherein the targeting molecule binds uPAR, EGFR, IGF-1R, orHER-2.
 9. The composition of claim 1, wherein the protease comprisesHEXXHXXGXXH (SEQ ID NO: 5) wherein X is any amino acid.
 10. Apharmaceutical composition comprising the composition of claim 1, and apharmaceutically acceptable excipient.
 11. A pharmaceutical compositionof claim 10 for use in the treatment of cancer.
 12. The pharmaceuticalcomposition of claim 10 comprising a second anti-cancer agent.
 13. Amethod of treating or preventing cancer comprising administering aneffective amount of a pharmaceutical composition of claim 10 to asubject in need thereof.
 14. The method of claim 13, wherein thepharmaceutical composition is administered in combination with a secondanti-cancer agent.
 15. A recombinantly produced polypeptide, wherein thepolypeptide comprising a human uPA sequence or segment thereofconfigured to bind urokinase plasminogen activator receptor and acatalytic domain of a protease polypeptide.
 16. A recombinant nucleicacid encoding the polypeptide of claim
 15. 17. A recombinant vectorcomprising a recombinant nucleic acid of claim
 16. 18. An expressionsystem configured to produce a recombinant polypeptide of claim 15comprising a vector of claim
 17. 19. The recombinantly producedpolypeptide of claim 15, wherein the human uPA sequence or segmentthereof configured to bind urokinase plasminogen activator receptorcomprises SEQ ID NO: 2 or a polypeptide with greater than 95% sequenceidentity thereto and the catalytic domain of a protease polypeptidecomprises SEQ ID NO: 3 or a polypeptide with greater than 95% sequenceidentity thereto.
 20. The recombinantly produced polypeptide of claim15, wherein the polypeptide comprises SEQ ID NO: 4 or a polypeptide withgreater than 95% sequence identity thereto.